1. Field of the Invention
The invention relates to methods for the mass spectrometric analysis of microbes from colonies on surfaces of nutrient media, particularly in a mass spectrometer with ionization by matrix-assisted laser desorption (MALDI).
2. Description of the Related Art
The routine, fast and error-free analysis of microorganisms plays an important role particularly in clinical and extra-clinical infection diagnostics, hygiene monitoring in hospitals or rivers and lakes used for swimming, food analysis, monitoring and control of biotechnological processes, and in microbiological research. The term microorganisms, here called microbes for short, describes all microscopically small organisms, for example unicellular fungi (e.g. yeasts), algae, or protozoae (e.g. plasmodia as malaria pathogens), although the focus of the identification work is usually on bacteria.
The identification of microbes means, in principle, determining their species and thus categorizing the microbes into the taxonomic hierarchy: domain (bacteria, archaea and eukaryotes), kingdom, phylum, class, order, family, genus, and species. In addition to taxonomic identification, the analysis of microbes can also comprise their characterization in terms of other properties, such as the pathogenicity of a microorganism (ability to cause disease), or the resistance of a microorganism against antibiotics.
For mass spectrometric identification methods, microbes taken from samples for analysis are usually cultured on nutrient media in Petri dishes to form colonies. The length of time between the samples for analysis being delivered to the analytical laboratory and the identification of the species is essentially dictated by the time needed for culturing, because the actual mass spectrometric determination takes only minutes. At present, this culturing process often takes between 18 and 24 hours. This is too long for many applications, particularly for applications in medical diagnostics. There is therefore an urgent need to significantly shorten the time required for the mass spectrometric identification, particularly to one working day.
With the methods currently used, the nutrient medium for the culture is usually contained in an agar in a Petri dish (agar plates), resulting in pure “isolates” in separated colonies. Agar is a gelatinous galactose polymer comprising much more than 90 percent water. The agar itself is indigestible and is attacked hardly at all by microbes. Since the microbes are mainly sampled manually at present, the colonies should have diameters of at least half a millimeter, better at least one millimeter, for reliable sampling of the microbes. To culture colonies of this size takes many hours, or sometimes even days, depending on the vigor of the microbes; for the clinically important species, the samples on agar plates are usually cultured for around 18 to 24 hours nowadays. If the colonies overlap or mix, isolated colonies are obtained in a second culture.
During the manual preparation of a MALDI sample, a small quantity of a selected colony is transferred from the surface of the nutrient medium onto a sample support; in practice this is often done with a wooden toothpick which is disposed of afterwards. The transferred microbes are then sprinkled with a strongly acidified solution of a conventional matrix substance (usually α-cyano-4-hydroxycinnamic acid, HCCA, or 2,5 dihydroxybenzoic acid, DHB) for a subsequent ionization by matrix-assisted laser desorption (MALDI). The acid (usually formic acid or trifluoroacetic acid) attacks the cell walls, which means that the organic solvent (usually acetonitrile) of the matrix solution can penetrate into the microbial cells and cause their weakened cell walls to burst by osmotic pressure. The destruction of the usually resilient cell walls is called “cell disruption;” cell disruption releases the soluble proteins from the cell. The sample is then dried by evaporating the solvent, causing the dissolved matrix material to crystallize. The released soluble proteins of the microbes, and also other substances of the cell to a small extent, are incorporated into the matrix crystals during the crystallization. This process produces a sample preparation on the sample support, which is called “MALDI sample” below.
The MALDI samples with the embedded analyte molecules are bombarded with focused UV-laser pulses of a few nanoseconds duration in a mass spectrometer, thus generating ions of the analyte molecules in the vaporization plasmas. These ions can then be separated from each other in the mass spectrometer according to the mass of the ions, and can be measured. Currently, simple time-of-flight mass spectrometers without a reflector are used for the mass spectrometric identification of microbes in order to achieve the highest sensitivity.
The mass spectrum is the intensity profile of the mass values of the analyte ions from the microbes. The ions here are predominantly protein ions, and the ions with the most useful information for identification have masses of approximately between 3,000 daltons and 15,000 daltons. In this method the protein ions are predominantly only singly charged (charge number z=1), which is why one can also simply talk about the mass m of the ions here, instead of always using the term “mass-to-charge ratio” m/z, as is actually necessary in mass spectrometry. The identification is carried out by similarity comparisons between the mass spectra acquired from the microbes and reference spectra from a reference library; see the document DE 10 2010 006 450 A1 (M. Kostrzewa), which also contains a detailed description of the mass spectrometric method. For medical applications, suitable reference libraries with reference mass spectra from several thousand microbe strains are now commercially available.
The mass spectrometric method for the identification is very robust; changes to the culture conditions or the preparation methods have hardly any effect on the identification results because practically only genetically defined proteins with genetically defined abundances are analyzed for each species. Around 60 to 85 percent of the proteins originate from the ribosomes, which comprise a fixed number of between 40 and 60 different protein molecules, the number depending on the species. Each bacterial cell contains several ten thousand identical ribosomes; cells of eukaryotes contain several hundred thousand. Thus the abundances of the measured proteins do not depend on the nutritional conditions or the maturity of the colony, as is the case with lipoproteins or fatty acids serving as energy stores, for example. The robustness of the method makes it possible to use microbes from very young, or mature or even ageing colonies for identification, and approximately the same identification results are achieved for these colonies.
To date the rule of thumb has been that around 105 microbes, at least, are required for preparing the MALDI sample on the sample plate in order to guarantee a reliable mass spectrometric identification of the microbes. This quantity is hardly discernible with the naked eye. Particularly suitable are quantities between 105 and 107 microbes. In the case of eukaryote cells with several hundred thousand ribosomes, usable mass spectra have been successfully obtained from individual cells. But for bacteria, whose hard cell walls require special cell disruption processes, it has so far only been possible in rare cases to produce mass spectra which are good enough for an identification from only 103 bacteria or less.
The mass spectrometric method of identification has proven to be extremely successful. It is very fast once culturing has been completed, and the certainty of correct identification is far greater than with the microbiological identification methods currently in use, as has been demonstrated in various studies.
The pursuit of automation has led to devices which replace manual transfer with machine transfer using a small inoculating rod. The Fraunhofer Institute for Factory Operation and Automation (Magdeburg/Germany) has developed a robot called “MiRob”, which can perform this task (cf.: O. Lange et al. (2008) “MIROB: automatic rapid identification of micro-organisms in high through-put”, Industrial Robot: An International Journal, Vol. 35 Iss: 4, pp. 311-315 or patent DE 10 2004 020 885 B2). The robot is manufactured as Mi Rob 300i by the company Mess-, Prüf- and Handling-Systeme GmbH, Reutlingen/Germany. patent DE 10 2004 020 885 B2, O. Lange et al.). As is the case with manual transfer, the microbes are transferred indirectly onto the mass spectrometric sample support by means of a tool, in this case an inoculating rod. Here too, the colonies should have a minimum diameter of 0.5 millimeters. The transfer tools used to date (toothpicks, inoculating rods) are designed to be used only once.
It would be desirable to have methods for the mass spectrometric analysis of microbes with which the time required from the delivery of samples to be investigated (sample for analysis) through to the identification is significantly shortened in comparison with current methods, preferably to one working day. The method should also be reliable and capable of automation, and should require little consumable material.